Recent advances in the pathogenesis and prevention strategies of dental calculus

Dental calculus severely affects the oral health of humans and animal pets. Calculus deposition affects the gingival appearance and causes inflammation. Failure to remove dental calculus from the dentition results in oral diseases such as periodontitis. Apart from adversely affecting oral health, some systemic diseases are closely related to dental calculus deposition. Hence, identifying the mechanisms of dental calculus formation helps protect oral and systemic health. A plethora of biological and physicochemical factors contribute to the physiological equilibrium in the oral cavity. Bacteria are an important part of the equation. Calculus formation commences when the bacterial equilibrium is broken. Bacteria accumulate locally and form biofilms on the tooth surface. The bacteria promote increases in local calcium and phosphorus concentrations, which triggers biomineralization and the development of dental calculus. Current treatments only help to relieve the symptoms caused by calculus deposition. These symptoms are prone to relapse if calculus removal is not under control. There is a need for a treatment regime that combines short-term and long-term goals in addressing calculus formation. The present review introduces the mechanisms of dental calculus formation, influencing factors, and the relationship between dental calculus and several systemic diseases. This is followed by the presentation of a conceptual solution for improving existing treatment strategies and minimizing recurrence.

examine the composition and function of oral microbial communities.Key oral pathogens have been identified from ancient dental calculus 7 .Furthermore, analyses of ancient dental calculus suggest that changes in bacterial diversity correlate with dietary shifts and variations in social environments 8 .
Formation and functions of bacterial biofilm and matrix.Bacterial biofilm consists of bacteria that are surrounded by a polymeric matrix produced by the bacteria 9 .The process of bacterial biofilm formation has been well investigated.Bacteria first attach reversibly to teeth through van der Waals forces and hydrophobic interactions.They use their pili to irreversibly attach to tooth surfaces, and form colonies to stabilize their attachment 10 .During the process, a self-produced matrix of extracellular polymeric substance (EPS) acts as a hard shell to protect the bacteria from external pressure 11 .The EPS has additional functions such as mediation of bacteria migration and provision of signaling targets.Accumulation of small bacterial clusters and bacterial layers subsequently results in the formation of plaque biofilm 12 .Channels and pores within the biofilm help the circulation of nutrition between the internal biofilm milieu and the exterior environment 13 .Bacteria present in dental plaque are divided into early colonizers and late colonizers.Early colonizers are typically facultative anaerobes and saccharolytic species that thrive on salivary mucins and other glycoproteins 3 .Late colonizers are usually proteolytic obligate anaerobes.The "bridge organism" between early and late colonizers is Fusobacterium nucleatum.This bacterium is capable of co-aggregating with other bacteria species 14 .Mature biofilms provide a platform for mineralization 15 .Among the plethora of bacteria present in plaque biofilms, Streptococcus sanguinis is the primary colonizer.Streptococcus mutans and Streptococcus gordonii co-aggregate over the primary colonizer via molecular interactions.The variety of bacterial species and interaction between distinct layers results in the formation of stable plaque biofilms 16 (Fig. 2).Changes within the bacteria also play an important role in mature biofilms.As a second messenger, intracellular cyclic di-guanosine monophosphate promotes surface attachment of biofilms.Reduction in intracellular cyclic di-guanosine monophosphate causes biofilm dispersion 17 .
The composition of the extracellular matrix also affects the status of plaque biofilms 18 .Compared with healthy teeth samples, teeth with dental calculus have more bacterial virulence proteins, which worsen the periodontal microenvironments.Glucosyltransferases produced by bacteria are released to the dental calculus matrix by exocytosis 19 .These enzymes catalyze the production of exopolysaccharides and promote bacteria adhesion to the surface of early biofilms 20 .Matrix macromolecules such as extracellular DNA (exDNA) are characteristic of mature biofilm in periodontitis.Extracellular DNA exists in the matrix in two forms: free DNA and extracellular DNA traps(ETs) (Fig. 3).Irrespective of the origin of exDNA, these molecules play a crucial role in initiating biofilm formation and sustaining the biofilm's three-dimensional structure 21,22 .The adhesion of S. mutans to hydrophobic surfaces and the viscoelasticity of biofilms are enhanced by exDNA.Under nutrient-limiting conditions, bacteria release exDNA enzymes that utilize exDNA as a surrogate source of nutrition 23 .Besides, Quorum sensing among biofilm bacteria, cyclic diguanylate, and small noncoding RNAs within the matrix regulatory network is involved in the regulation of biofilm formation 24 .
Biofilm formation on dental implants and teeth follows a similar pattern.However, there are a few differences due to the implant materials   used.These materials have a lower capacity for albumin absorption compared with natural teeth.This property results in reduced biofilm formation around implants.The process of biofilm formation begins on the implant surface ~30 min after exposure to the oral environment.In contrast, biofilms around natural teeth form within minutes 25 .Initially, implants are primarily colonized by gram-positive cocci, rods, and actinomyces species, which attach to salivary agglutinins and proline-rich proteins.These early colonizers pave the way for subsequent attachment by late colonizers such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum.The stepwise colonization by bacteria facilitates the development of biofilm and escalates its pathogenicity over time 26 .In addition, microgaps ranging from 2.5-60 μm in the implant-abutment interface impair the seal, allowing bacteria to infiltrate and form biofilms within the implant's inner threads 27 .Biofilms contribute to the formation of dental calculus, which in turn deteriorates the surface of the implant and increases its roughness.This roughness fosters further bacterial re-colonization, hindering the reattachment and proliferation of host cells 28 .
Mechanism of dental calculus biofilm mineralization.Mature plaque biofilms formed on the surfaces of teeth and dentures provide platforms for mineralization.Plaque bacteria are engaged in different metabolic processes that interact with inorganic minerals to produce mineral precipitates in dental calculus.The most common minerals deposited by bacteria are calcium carbonate and calcium phosphate 29 .The most frequent calcium phosphate crystalline phases found in dental calculus are hydroxyapatite, octacalcium phosphate, and whitlockite 30 .However, the local concentrations of calcium and phosphate ions are not high enough to cause spontaneous precipitation 31 .Therefore, bacteria play an important role in mineral deposition.It is necessary to understand the mineralization mechanism of the interaction between bacteria and minerals.Bacteria release different enzymes during the mineralization process.Recently, the release of alkaline phosphatase (ALP) was reported to be associated with periodontal inflammatory responses 32 .ALP has also been identified as a hallmark enzyme in periodontitis.Microorganisms in the dental biofilm induce the death of epithelial cells.Destruction of the epithelial barrier induces infected epithelial cells and immune cells to produce pro-inflammatory cytokines and chemokines, such as IL-1β, IL-6, and IL-8 33 .The pro-inflammatory cytokines recruit inflammatory cells and initiate the subsequent inflammatory response.The ALP is released by bacteria 34 .Bacterial ALPs are found extracellularly, bonding on the surface of the bacteria.Alkaline phosphatase has the ability to dephosphorylate teichoic acid, a molecule that plays a key role in bacterial colonization on teeth.With the release of phosphate ions, calcium ions may become supersaturated 35 .It is also reported that ALP regulated biofilm formation by promoting aerobic pathways 35 .This shows the importance of ALP in the mineralization of biofilm.
In addition, positively-charged polycationic compounds also induce dental calculus mineralizati on36 .For instance, streptococcal bacterial protein is rich in positively-charged amino acids.It acts as a substrate for the nucleation and growth of inorganic mineral particles 37 .Environmental changes also affect dental calculus mineralization.Bacteria produce enzymes that influence the environment and regulate calcium and phosphorus content 38 .Ureolytic bacteria hydrolyze salivary and dietary urea via the enzyme urease to produce ammonia and carbon dioxide, increasing the pH value.Thus, calcium phosphate saturation increases in plaque fluid and accelerates the formation of dental calculus 17 .
Effects of physicochemical factors on bacteria.The relationship between the oral microbiota and the oral physicochemical environment changes dynamically.This relationship may evolve from a healthy symbiotic relationship to pathological dysbiosis 39 .Studying the influence of oral physicochemical factors on bacterial status can help elucidate the mechanisms of calculus formation.In addition, changes in physical and chemical factors may also cause changes in supragingival or subgingival plaque microflora (Fig. 4).

Immune factors
Periodontal crevices and pockets are filled with gingival crevicular fluid.The latter is rich in immune cells.Under the stimulus of a biofilm, neutrophils 40 , and macrophages 41 are recruited to the pocket site and activated.Neutrophil extracellular traps (NETs), secreted by neutrophils, play a dual role in both protecting the host from infections and facilitating the mineralization of dental calculus.These NETs are capable of capturing pathogens, thereby limiting infection 42 .There is substantial evidence suggesting that NETs are instrumental in the formation of gallstones, salivary stones, and monosodium urate crystals [43][44][45] .A recent study revealed that NETs-DNA acts as nucleation sites for pathological mineralization of dental calculus.The study demonstrated that bacteria within the mature biofilms secrete histone-like proteins that alter the chiral structure of NETs-DNA from the B-form to the Z-form.This transformation reduces the susceptibility of NETs to degradation by deoxyribonuclease I, thereby enhancing their ability to induce mineralization 46 (Fig. 5).Bacteria and mineralized crystallites further recruit and polarize macrophages into classically-activated subtypes 47 .The activated macrophages release exosome-like calcifying extracellular vesicles.These vesicles are rich in calcium ions and are capable of mineralization.During the formation of calculus, neutrophils act more promptly than macrophages 48 .Hence, it is important to reduce the contribution of neutrophils to mineralization.Targeted inhibition of extracellular nuclease released by bacteria may enhance the efficiency of neutrophils and delay the mineralization of bacterial biofilms.

Host factors
Lifestyles have significant effects on calcium phosphate saturation and bacterial colonization.Tobacco smoking induces higher salivary calcium contents which increase the formation of calculus [49][50][51] .A carbohydratedominated diet may induce dental calculus by promoting bacterial colonization on the plaque53.Sucrose is the most widely consumed dietary carbohydrate.This sugar modifies the local microenvironment, and is closely associated with increased dysbiosis in oral biofilms 52 .Replacing refined carbohydrates with foods high in fat and vitamin D could lessen bacterial adhesion to teeth 53 .However, it is necessary to ascertain whether individuals aiming to mitigate dental calculus find such dietary substitutions acceptable.

Novel prevention and treatment strategies for dental calculus
Conventional methods of dental calculus prevention and treatment are brushing, scaling, and root planning (SRP).Improving oral hygiene by brushing or using chlorhexidine mouth rinses is the most economical and convenient way to prevent the development of calculus 54 .The use of SRP polishes the tooth surface to prevent the re-deposition of biofilms and dental calculi 55 .Given the main pathogenic factors of periodontitis are dental calculus and plaque that are attached to the root surface, this oral disease may be treated by subgingival scaling.This treatment reduces the depth of periodontal pockets and renders the root surface smooth.The condition that is favorable to facilitate the reattachment of the gingiva to the root surface is created 56 .These changes reduce the chance for bacteria to attach.Apart from physical removal techniques, chemical agents such as pyrophosphates play a crucial role in dental calculus management.Pyrophosphates disrupt bacterial growth by hindering the biosynthesis of the bacterial cell wall, which obstructs lipid transport essential for cell wall integrity 55 .These compounds also lower calcium and phosphorus concentrations in the periodontal microenvironment.Consequently, pyrophosphates contribute to reducing the essential components required for bacterial mineralization 57 .This helps to prevent dental calculus formation.
Innovative treatment modalities for dental calculus have emerged in recent years.These include laser technology, new kinds of toothpaste, and natural plant-derived compounds 58,59 .In addition, vaccines and nanotechnology offer unique perspectives for the control of dental calculus.
Physical intervention is a common approach to preventing and treating dental calculus.Ultrasound relies on cavitation and acoustic microstreaming to achieve cleaning 60 .Shockwaves generated by lasers alter biofilm permeability to enable easier elimination of pathogenic bacteria 61 .Calculus removal with lasers leaves no residual calculi, which is better when compared with conventional manual instruments 62 .However, the use of erbium-doped yttrium/aluminum/garnet laser and erbium-chromium-doped yttrium/scandium/gallium/garnet laser results in rougher root surfaces 56 .A combination of SRP and erbium laser is the solution to solve the problem.It removes residual debris from the root surface with little or no adverse thermal damage on the root surface 63 .Thermal damage, especially increased temperature within the periodontium, is considered a potential hazard to the dental pulp and periodontal tissues 64 .Thus, the advantage of laser technology for calculus removal is relatively limited due to the risk of thermal damage 59 .The effectiveness of the erbium laser and SRP in non-surgical periodontal treatment needs further evaluation.
Damage to patients' enamel surfaces with manual and ultrasonic scalers has been a problem that plagued clinicians.To address this issue, scientists developed a novel actuator-driven pulsed water jet (ADPJ) system 65 .The system is capable of selectively removing materials based on their hardness.Because of the different hardness of teeth and dental calculus, the ADPJ system removes dental calculus with appropriate jet pressure without damaging the enamel surface of the tooth 66 .The ADPJ system was shown to significantly reduce the amount of dental calculus, and the removal rate was dependent on the applied voltage.No enamel damage was observed on the tooth surface after using the ADPJ system when compared to conventional scalers.
Aragonite (a calcium carbonate mineral phase) can be extracted from animals such as cuttlefish.It is an abrasive with a hardness that facilitates the removal of dental calculus 67 .Scientists tested the effectiveness of aragonite toothpaste derived from squid to remove dental calculus 53 .The aragonite toothpaste had a good effect in removing calculus, preventing calculus formation, and improving gingival health.The aragonite toothpaste dud bit causes any enamel damage 53 .
Vaccines have been anticipated to improve the prevention and treatment of periodontal disease.However, the immunopathological complexity of periodontal disease complicates the development of periodontal vaccines 57 .Studies suggest that successful periodontal vaccines may require mucosal delivery and polyvalent approaches.This strategy requires clinical trials for validation of the treatment outcomes 57 .
Other scientists utilized nanoparticles and biological agents to prevent the formation of bacterial biofilms.Metal and metal oxide nanoparticles have broad application prospects due to their small size, large specific surface area, and antibacterial properties 68 .The incorporation of silver and gold nanoparticles in toothbrushes enhances the mechanical control of dental plaque 69 .Platinum nanoparticles have many outstanding features.They provide powerful mechanical control and have the potential to degrade proteins and periodontal disease-associated inflammatory factors, such as lipopolysaccharides 70 .The specific mechanism of nanoparticles on bacteria is shown in Fig. 6.
Factors such as the size of the nanoparticles can also affect the efficacy of calculus removal.Other novel methods for preventing calculus formation are in their infancy of development (Fig. 7).
Active patient participation and maintenance of good oral hygiene are essential to prevent plaque and biofilm formation.The development of mouthwash or toothpaste may be a promising avenue to facilitate the prevention of calculus formation.These products exhibit low toxicity, high biosafety, and exceptional efficacy.By continuously exploring novel treatment modalities, the incidence of dental calculus will be greatly reduced.This also reduces the potential harm of dental calculus on systemic diseases.

Outlook
Periodontal diseases caused by dental calculus affect both oral and systemic health.The present review systematically describes the mechanisms of calculus formation in the human oral environment.Considerations need to be given to both bacterial and environmental factors that cause calculus deposition on tooth surfaces.The mechanisms of plaque biofilm formation as well as the alteration of bacterial types during the formation of biofilm and calculus are also highlighted.In addition, the influence of the inflammatory state of the body and the physicochemical state of the saliva on the formation of dental calculus has also been demonstrated.To alleviate clinical problems, new treatment methods for calculus removal have emerged in recent years.However, the interconnection between dental calculus formation and other systemic diseases is not completely understood.In the future, healthcare workers need to objectively address multiple factors such as age, gender, race, and religious belief.Systematic classification of patients as well as the condition of diseases will be used to design individualized treatment.At the same time, doctors need to raise awareness of the importance of oral hygiene and the early diagnosis of gingival and periodontal problems.To achieve this goal, preventive and curative oral health policies adapted to patients' diverse living environments need to be planned and implemented, with resources, goals, and priorities identified.

Fig. 1 | 1 Fig. 2 |
Fig. 1 | Pathogenesis of dental calculus involves bacteria and matrix mineralization.Bacteria induce destruction of biological macromolecules, tissue abnormalities, and inflammatory responses.Mineralized matrix releases heavy metal ions and causes inflammatory responses.(ROS reactive oxygen species, M1 classically activated macrophages, M2 alternatively activated macrophages, Th cells T helper cells, TNF-a tumor necrosis factor-a) (The figure does not contain any third-party material, the figure and each of the elements in the figure were created by the authors).

Fig. 3 |
Fig. 3 | The origin of exDNA.a Free DNA is derived from multiple sources.These molecules are produced mainly by endogenous pathways, including active secretion, erythrocyte nucleation, necrosis, and degradation of ETs from neutrophils.b Three types of extracellular vesicles carry exDNA 22 .Figure reproduced with permission from the publisher.

Fig. 4 |
Fig. 4 | Physical and chemical factors present in supragingival or subgingival plaque.The types of bacteria located in the supragingival and subgingival plaques are different.The physicochemical factors affecting the bacteria in the two sites are also different 72 .This schematic is reproduced with permission from the publisher.

Fig. 5 |
Fig. 5 | Formation of dental calculus in the presence of NETs. a Bacterial biofilms trigger the transformation of NET-DNA into DNase-I-resistant NET-Z-DNA.b NET-DNA plays a role in regulating ectopic mineralization in vitro.c The mineralization mechanism of dental calculus involving NETs 46 .a, b, and c are reproduced with permission from the publisher.

Fig. 6 |
Fig. 6 | Mechanisms of nanoparticle-mediated antimicrobial and anti-biofilm activities against oral bacteria.a The inhibition of glucosyl transferase by nanoparticles results in reduced exopolysaccharide production and biofilm formation.

Fig. 7 |
Fig. 7 | Nanoparticle-mediated antimicrobial and anti-biofilm activities against oral bacteria.a Size and shape, surface, and interior properties of nanoparticles are important for their use in biofilm-infection control 73 .b Effect of antibiotics on biofilms.Other strategies employed for the treatment of biofilms include the use of antimicrobial peptides (AMP), bio-surfactants (rhamnolipid), phage particles, cheat cells, and different EPS-disrupting enzymes 74 .a and b are reproduced with permission from the publisher.

Table 1 |
Secondary diseases and their pathogenic factors due to dental calculus